The Inventor has developed and successfully applied a metabolic model of phenylalanine (Phe) metabolism to treatment development for neurological disorders, both to those seen only in psychiatric patients, and to those seen in the general population (U.S. Pat. No. 5,393,784; U.S. Pat. No. 5,670,539, allowed application U.S. application Ser. No. 08/545,095). The treatment method relates to a medical food product which has been clinically proven to reduce the availability of Phe to the central nervous system. The instant invention applies the Phe metabolism model to define and develop treatments to the genetics of psychotic, mood and personality disorders based on the results of (a) DNA analysis of the phenylalanine hydroxylase (PAH) gene, and (b) Phe dosing studies, in 124 patients with these disorders.
The PAH enzyme catalyzes the conversion of Phe to tyrosine (precursor of the amine neurotransmitter, dopamine), and deficient activity of this enzyme leads to increased plasma levels of Phe. Phe, as an indirect precursor of dopamine and noradrenaline, supports their synthesis at low plasma concentrations while at higher concentrations, it inhibits the synthesis of these two neurotransmitters in addition to serotonin. Thus plasma Phe levels play an important role in modulating neurotransmitter synthesis.
A model for Phe metabolism is valuable in the study of psychotic, mood and personality disorders (a) because of the role of Phe in amine neurotransmitter synthesis, (b) the suspected role of neurotransmitter function in these disorders, and (c) the fact that the limited treatment success that has been seen in these disorders is from agents that regulate neurotransmitter synthesis. Further, the acute sensitivity of the brain to higher than normal levels of Phe and its metabolites that occurs with deficiencies of PAH activity is well exemplified by the mental retardation, seizures, spasticity and EEG irregularities seen in phenylketonuria, a hyperphenylalanemia caused by deficient or absent PAH activity.
The psychotic disorders extract the greatest cost to the patient, family members, and to society at large. The most prevalent of the psychotic disorders both in the DNA study subject sample of the present invention and the general population is the disorder of schizophrenia. Schizophrenia is the most serious and the most treatment resistant of the disorders. It is heterogeneous in expression, etiology, and pathophysiology, with sex often being a defining variable. The disorder is known to have a complex pattern of inheritance indicative of interactions among multiple genes and environmental factors and has been difficult to analyze at a genetic level. The present invention relates to methods of identifying and methods of treating the psychotic disorder of schizophrenia, though the invention is applicable to the entire group of psychotic, mood and personality disorders.
DNA analyses of the PAH gene in the study sample have revealed significant associations between the disorder of schizophrenia and both a novel missense mutation (K274E) and novel polymorphism (L321L). There was a statistical association between the presence of these two variants in the study sample, and all persons with either of these variants were of African-American ethnicity. The K274E missense mutation results in a lysine to glutamic acid substitution at amino acid 274, the substitution of which may cause changes in the physical and chemical properties of the PAH protein. The physiological effect of the mutation may be also be relevant to schizophrenia since the mutation is located in a region considered to be involved in the binding of tetrahydrobiopterin (BH4). BH4 is the cofactor for Phe, tyrosine and tryptophan hydroxylases, all of which are required for the synthesis of the amine neurotransmitters dopamine, noradrenaline, and serotonin. These results suggest that the mutation may be relevant to the pathophysiology of schizophrenia.
Analysis of two-hour post Phe dose plasma Phe and tyrosine levels in the DNA study sample demonstrates (a) significantly higher Phe plasma levels, and (b) significantly less conversion to tyrosine, for those with the K274E mutation, indicating reduced catabolism of Phe for that group. In addition, differences in plasma levels for neopterin, biopterin and neopterin/biopterin ratio were observed between patients with and without the mutation, suggesting that increased synthesis of BH4 may be associated with the K274E mutation. The increase in BH4 synthesis may be a homeostatic response to reduced levels of PAH activity.
The effect of the novel L321L polymorphism on PAH enzyme activity is not known although the L321L polymorphism was found to be associated with schizophrenia and the K274E mutation. Studies of the variant may include investigations of DNA methylation and/or RNA splicing, stability or utilization. The significance of the L321L polymorphism is supported by the observation that patients with this polymorphism had significantly higher two-hour post Phe dose Phe plasma levels, and significantly less conversion to tyrosine, indicating reduced catabolism of Phe.
The nationally stated goal of the field of genetic research in psychotic, mood and personality disorders is to identify mutations that confer susceptibility to illness and that have predictable and understandable pathophysiological effects that may be related to these disorders. The present invention is a unique, hypothesis-driven approach toward meeting the goals of the field with (a) the discovery of a novel missense mutation and a novel polymorphism on a gene related to neurotransmitter synthesis in persons with psychotic, mood and personality disorders, (b) the discovery of a statistically significant association between the presence of these two variants, (c) the discovery of a statistically significant association between these two novel variants and the disorder of schizophrenia, (d) the discovery of a plasma dose response which supports significant physiological effects for the K274E mutation and L321L polymorphism and implicates these variants in the reduced catabolism of Phe, and (e) the discovery of a significant effect for the mutation in the increased synthesis of BH4. Further, the ethnic homogeneity for the two novel PAH variants associated with schizophrenia improves the prospects of making a meaningful contribution to the understanding the genetics of the complex disorder of schizophrenia. Ethnic considerations are of critical importance in genetic studies. For example, in patients with schizophrenia, significant differences were observed between persons of African and European descent with respect to heterozygosity and the number of alleles per marker, whereas in patients with Alzheimer""s Disease, the APOE-e4 allele is consistently associated with a greater risk for the disease in Caucasians but not in African-Americans.
Treatment of the disorder of phenylketonuria is an example where early detection and therapeutic intervention has made a significant impact on the overall cost to society. The deleterious central nervous system effects of that disorder, also caused by mutations and polymorphisms on the PAH gene, have been significantly diminished in the western world because of newborn screening of Phe plasma levels. Infants with high levels of Phe are placed on a Phe-restricted diet, and as a direct consequence of such dietary restriction the central nervous system is protected from severe consequences of high plasma Phe such as mental retardation. The association of the novel K274E mutation and L321L polymorphism with schizophrenia in African-Americans in combination with pedigree analysis may also have enormous public health benefits with respect to the treatment of the disorder schizophrenia.
The phenylalanine hydroxylase enzyme catalyzes the conversion of the large neutral amino acid phenylalanine (Phe) to tyrosine, which is the rate-limiting step in the catabolism of Phe. The brain is highly sensitive to levels of Phe, and deficiencies in the PAH enzyme may result in excess levels of Phe or hyperphenylalanemia. Deficiencies in PAH enzyme activity may range from classical PKU and its potential for severe central nervous system dysfunction (mental retardation), to moderate elevations in plasma Phe with no known clinical consequences. A deficiency in PAH enzyme activity is the most common cause of hyperphenylalanemia, with 99% of the mutant alleles mapping to the PAH gene and the remainder mapping to several genes involved in the synthesis and recycling of tetrahydrobiopterin (BH4), the cofactor in the hydroxylation reaction (Scriver C R, xe2x80x9cWhatever happened to PKU?xe2x80x9d, Clin. Biochem., 1995, 28: 137-144). The human PAH gene spans 90 kb, is comprised of 13 exons, and has been localized to chromosome 12q24.1 (Lidsky A S et al., xe2x80x9cRegional mapping of thephenylalanine hydroxylase gene and the phenylketonuria locus in the human genomexe2x80x9d, Proc Natl Acad Sci USA, 1985, 82:6221-6225; DiLela A G et al., xe2x80x9cMolecular structure and polymorph map of the human phenylalanine hydroxylase genexe2x80x9d, Biochemistry, 1986, 25:743-749). Structure/function analyses have identified a central catalytic domain which contains sites for substrate, iron and BH4 cofactor binding, an N-terminal region with regulatory properties, and a C-terminal domain involved with intersubunit binding (Hufton S E et al., xe2x80x9cStructure and function of the aromatic amino acid hydroxylasesxe2x80x9d, Biochem J, 1995, 311:353-366; Waters P J et al., xe2x80x9cIn vitro expression analysis of mutations in phenylalanine hydroxylase: linking genotype to phenotype and structure to functionxe2x80x9d, Hum Mutat, 1998, 11:4-17).
Although some studies have suggested that patients with PKU and heterozygotes for PKU might have an increased susceptibility for psychiatric disorders such as schizophrenia (Thompson J H, xe2x80x9cRelatives of phenylketonuric patientsxe2x80x9d, J Ment. Defic. Res. 1957; 1:67-78; Kuznersova L I, xe2x80x9cFrequency and phenotypic manifestations of schizophrenia in the patients with phenylketonuriaxe2x80x9d, Sov. Genet. 1974:554-555) this remains a topic of considerable controversy. Several investigations have found that there is no statistical evidence to demonstrate that the frequency of mental illness in PKU patients and their families exceeds levels in control populations (Perry T L et al., xe2x80x9cThe incidence of mental illness in relatives of individuals suffering from phenylketonuria or mongolismxe2x80x9d, J. Psychiatr. Res. 1966; 4:51-57; Blumenthal M D, xe2x80x9cMental illness in parents of phenylketonuric children:xe2x80x9d, J. Psychiatr. Res. 1967; 5:59-74; Larson C A and Nyman G E, xe2x80x9cPhenylketonuria: mental illness in heterozygotesxe2x80x9d, Psychiatr. Clin. 1968; 1:367-374; Pietz J et al., xe2x80x9cPsychiatric disorders in adult patients with early-treated phenylketonuriaxe2x80x9d, Pediatrics, 1997, 99:345-350).
Investigations of Phe metabolism in psychiatric disorders are few and the findings are equivocal. Some studies have indicated that schizophrenic patients have higher levels of plasma Phe than control subjects (Poisner A M, xe2x80x9cSerum phenylalanine in schizophrenia; biochemical genetic aspectsxe2x80x9d, J. Nerv. Ment. Dis. 131:74-76, 1960; Bjerkenstedt et al., xe2x80x9cPlasma amino acids in relation to cerebrospinal fluid monoamine metabolites in schizophrenic patients and healthy controlsxe2x80x9d, Br. J. Psychiatry, 1985, 147:276-282; Rao M L et al., xe2x80x9cSerum amino acids, central monoamines, and hormones in drug-naive, drug-free, and neuroleptic-treated schizophrenic patients and healthy subjectsxe2x80x9d, Psychiatry Res. 1990, 34:243-257), while another reported no significant difference between the two groups (Szymanski H V et al., xe2x80x9cPlasma phenylethylamine and phenylalanine in chronic schizophrenic patientsxe2x80x9d, Biol. Psychiatry 1987; 22:194-198). The results for another index of Phe metabolism (the level of plasma Phe following a Phe challenge) have been similarly ambiguous, with a report from one group of investigators that post-challenge Phe levels were increased in schizophrenic patients compared to controls, which they subsequently failed to replicate (Wyatt R J et al., xe2x80x9cPhenylethylmine (PEA) and chronic schizophreniaxe2x80x9d, Catecholamines: Basic and Clinical Frontiers, Usdin K., Kopin I J, Barchas J D, eds. New York: Raven Press, 1979; 1833-1835; Potkin S G et al., xe2x80x9cPlasma phenylalanine, tyrosine, and tryptophan in schizophreniaxe2x80x9d Arch. Gen. Psychiatry 1983; 40: 749-752).
Another aspect of the studies on Phe metabolism in schizophrenia have focused on phenylethylamine, which is produced in excess by a decarboxylase pathway in the absence or impairment of PAH activity. Phenylethylamine has been proposed to act as a neuromodulator via serotonin receptors (Boulton A A et al., xe2x80x9cPhenylethylamine in the CNS: effects of monoamine oxidase inhibiting drugs, deuterium substitution and leisons and its role in the neuromodulation of catecholaminergic neurotransmissionxe2x80x9d, J. Neural Transmission Suppl. 1990; 29: 119-129, Sloviter R S et al., xe2x80x9cSerotonergic properties of b-phenethylamine in ratsxe2x80x9d, Neuropharmacology 1981; 20: 1067-1072) and is of interest in schizophrenia research since it is a chemical congener of amphetamine, abuse of which produces a psychosis resembling paranoid schizophrenia (Snyder S H, xe2x80x9cAmphetamine psychosis: a xe2x80x9cmodelxe2x80x9d schizophrenia mediated by catecholaminesxe2x80x9d, Am J. Psychiatry 1973; 130: 61-67). Investigators have reported differences between schizophrenic patients and control subjects including differences between groups of schizophrenic subtypes, as measured by the level of phenylethylamine in body fluids (Szymanski H V et al., xe2x80x9cPlasma phenylethylamine and phenylalanine in chronic schizophrenic patientsxe2x80x9d, Biol Psychiatry 1987; 22: 194-198.69; Potkin S G et al., xe2x80x9cPhenylethylamine (PEA) and phenylacetic acid (PAA) in the urine of chronic schizophrenic patients and controlsxe2x80x9d, Psychopharmacol Bull 1980; 16-:52-54; Jeste D V et al., xe2x80x9cCross-cultural study of a biochemical abnormality in paranoid schizophreniaxe2x80x9d, Psychiatry Res. 1981; 5: 341-352; Yoshimoto S et al., xe2x80x9cUrinary trace amine excretion and platelet monoamine oxidase activity in schizophreniaxe2x80x9d, Psyciatry Res. 1897; 21:229-236; O""Reilly R et al., xe2x80x9cPlasma phenylethylamine in schizophrenic patientsxe2x80x9d, Biol. Psychiatry 1991; 30:145-150; Fischer E et al., xe2x80x9cUrinary elimination of phenethylaminexe2x80x9d, Biol Psychiatry 1972; 5: 139-147). These studies on phenylethylamine, though more numerous than those on Phe, have been considered particularly problematic because of the trace amine status of phenylethylamine (small amounts, rapid transit through systems, unstable) which has resulted in technological difficulties in measurement of the amino and lack of conformity in values across studies.
The study of Phe metabolism is the most limited in psychiatric disorders other than schizophrenia. In patients with endogenous depression, unipolar depression or bipolar syndrome, the response to a Phe challenge was indistinguishable from that in control subjects (Pratt R T C et al., xe2x80x9cPhenylalanine tolerance in endogenous depressionxe2x80x9d, Brit. J. Psychiat., 1963, 109:624-628; Targum S D et al., xe2x80x9cScreening for PKU heterozygosity in bipolar affectively ill patientsxe2x80x9d, Biol. Psych., 1979, 14:651-655; Gardos G et al., xe2x80x9cThe acute effects of a loading dose of phenylalanine in unipolar depressed patients with and without tardive dyskinesiaxe2x80x9d, Neuropsychopharmacology, 1992, 6:241-247).
Only two investigations have examined the PAH gene and schizophrenia and both have produced negative findings. The first of these screened for two specific mutations in the PAH gene (the putative null mutations R408W and IVS 12nt1) in schizophrenics and normal controls. None of the schizophrenic subjects in the study were found to have these variants. It was thus concluded that neither of these genetic polymorphisms were associated with a predisposition to schizophrenia (Sobell J L et al., xe2x80x9cNovel association approach for determining the genetic predisposition to schizophrenia: case-control resource and testing of a candidate genexe2x80x9d, Am. J. Med. Genet. 1993; 48:28-35). The second study was a genome-wide search for schizophrenia susceptibility genes. The investigators failed to find a significant association between PAH and disease susceptibility (Shaw S H et al., xe2x80x9cA genome-wide search for schizophrenia susceptibility genesxe2x80x9d, Am. J. Med. Genet. 1998; 81:364-376).
No other studies have found an association between the PAH locus on chromosome 12 in reference to psychiatric disorders. One publication describes an investigation of the relationship between schizophrenia and the phospholipase-A2 gene, the result of which was negative (Psychiatr Genet. 1995, 5:177-80; O-Malley M P et al. Linkage analysis between schizophrenia and the Darier""s disease regionon 12q, Psychiatr Genet. 1996: 6:187-90).
The psychotic disorders are the most serious of the psychiatric illnesses and make up the bulk of the patients in the public sector extracting a major economic cost from society. Patients afflicted with psychosis suffer from symptoms such as delusions, hallucinations, disorganized speech and grossly disorganized or catatonic behavior. The disorders in this category are schizophrenia, schizophreniform disorder, schizoaffective disorder, brief psychotic disorder, delusional disorder, shared psychotic disorder, psychotic disorder due to a general medical condition, substance-induced psychotic disorder, psychotic disorder not otherwise specified (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Washington, D.C., American Psychiatric Association, 1994).
The most prevalent of these disorders is schizophrenia having a lifetime prevalence ranging from 0.5% to 1%, the particular symptoms of which are the following (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, D.C., American Psychiatric Association, 1994):
1. Delusionsxe2x80x94are erroneous beliefs that usually involve a misinterpretation of perceptions or experiences. Presecutory delusions are the most common; the person believes he or she is being tormented, followed, tricked, spied on, or subjected to ridicule. Referential delusions are also common; the person believes that certain gestures, comments, passages from books, newspapers, song lyrics or other environmental cues are specifically directed at him or her. Bizarre delusions are especially characteristic of schizophrenia. An example of a bizarre delusion is a person""s belief that a stranger has removed his or her internal organs and has replaced them with someone else""s organs without leaving any wounds or scars.
2. Hallucinationsxe2x80x94auditory hallucinations are the most common and are experienced as voices that are perceived as distinct from the person""s own thoughts. Pejorative or threatening voices are the most common. Most characteristic of schizophrenia are two or more voices conversing with one another or voices maintaining a running commentary on the person""s thoughts or behaviors.
3. Disorganized thinkingxe2x80x94formal thought disorder, loosening of associations; as manifested by speech that is disorganized enough to substantially impair effective communication; such as, derailment where the person slips off the track of a thought, answers to questions may be completely unrelated, speech may be so severely disorganized that it is incomprehensible as resembling a word salad.
4. Grossly disorganized behaviorxe2x80x94problems may be seen in any form of goal-directed behavior, leading to problems performing activities of daily living. For instance, the person may be markedly disheveled, dress in an unusual manner (e.g., multiple overcoats on a hot day), show inappropriate sexual behavior (e.g., public masturbation), show childlike silliness, show unpredictable and untriggered agitation.
5. Catatonic motor behaviorsxe2x80x94a marked decrease of reactivity to the environment which can reach to stupor, maintaining a rigid posture and resisting efforts to be moved, inappropriate or bizarre postures, or purposeless and unstimulated excessive motor activity.
6. Negative symptomsxe2x80x94persons face appears immobile and unresponsive with poor eye contact and reduced body language, speech is characterized by brief, laconic, empty replies, person has an inability to initiate and persist in goal-directed activities, and will sit for long periods of time and show no interest in participating in work or social activities.
The disorders in the category of Mood Disorders are; Major Depressive Disorder, Dysthymic Disorder, Depressive Disorder Not Otherwise Specified, Bipolar I Disorder, Bipolar II Disorder, Cyclothymic Disorder, Bipolar Disorder Not Otherwise Specified, Mood Disorder Due to a General Medical Condition, Substance-Induced Mood Disorder, Mood-Disorder Not Otherwise Specified (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, D.C., American Psychiatric Association, 1994).
The disorders in the category of Personality Disorders are; Paranoid Personality Disorder, Schizoid Personality Disorder, Schizotypal Personality Disorder, Antisocial Personality Disorder, Borderline Personality Disorder, Histrionic Personality Disorder, Narcissistic Personality Disorder, Avoidant Personality Disorder, Dependent Personality Disorder, Obsessive-Compulsive Personality Disorder, Personality Disorder NOS (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Washington, D.C., American Psychiatric Association, 1994).
It was through the results of several independent studies in these patient groups, that the Phe metabolic model was developed and which forms the background of the invention. The model is based on the following established criteria in relation to the role of Phe in neurotransmitter synthesis.
The main factors determining the entry of Phe into the brain are its concentration in plasma, the concentration of the competing large neutral amino acids in plasma, and the activity of the blood-brain barrier transport system or L-system. The L-system, which competitively mediates the bi-directional flux of the neutral amino acids between the blood and brain, has a preferential affinity for the large neutral amino acids, with Phe having the highest affinity among them (Daniel P M et al. xe2x80x9cAmino acid precursors of monoamine neurotransmitters and some factors influencing their supply to the brainxe2x80x9d Psychol. Med. 1976; 6: 277-286; Pardridge W M and Choi T B, xe2x80x9cNeutral amino acid transport at the human blood-brain barrierxe2x80x9d, Federation Proceedings, 1986, 45: 2073-2078). Due to the competition between amino acids for brain entry, higher concentrations of plasma Phe can result in lower brain availability of tyrosine and tryptophan. These latter amino acids serve as precursors in the synthesis of the neurotransmitters dopamine, serotonin and noradrenaline, all of which have been implicated in psychotic, mood and personality disorders. Further, an inhibitory effect of Phe and its metabolites (such as phenylethylamine and phenylacetic acid) on the activities of tyrosine hydroxylase, tryptophan hydroxylase, and DOPA decarboxylase further decreases the synthesis of dopamine, noradrenaline and serotonin (Caballero B and Wurtman R J, xe2x80x9cControl of plasma phenylalanine levelsxe2x80x9d Ch. 1, In: Wurtman R J and Ritter-Walker, E, eds. Dietary Phenylalanine and Brain Function. Boston: Birkhauser, 1988; 3-12; Lehnert H and Wurtman R J, xe2x80x9cAmino acid control of neurotransmitter synthesis and release: physiological and clinical implicationsxe2x80x9d Psychotherapy and Psychosomatics, 1993; 60: 18-32; Maher T, xe2x80x9cEffects of phenylalanine on the synthesis, release and function of catecholaminergic systemsxe2x80x9d in Amino Acids in Psychiatric Disease. Washington: American Psychiatric Press, Inc., 1990; 131-142; Scriver C R and Rosenberg L E, xe2x80x9cPhenylalaninexe2x80x9d Ch. 15, In: Schaffer Al, eds. Amino Acid Metabolism and its Disorders. Philadelphia, 1973; 290-337). Thus the integrity of the Phe metabolic system is critically important in the function of neurotransmitter systems thought to be involved in these disorders. Higher plasma Phe levels have been shown to be significantly associated with lower central nervous system levels of tyrosine and trytophan in a sample of schizophrenics (Bjerkenstedt L et al. xe2x80x9cPlasma amino acids in relation to cerebrospinal fluid monoamine metabolites in schizophrenic patients and healthy controlsxe2x80x9d Br. J. Psychiatry, 1985; 147: 276-282).
The Phe metabolic model was initially developed to allow for the development of treatment strategies for a neurological disorder, tardive dyskinesia (TD). This disorder is a side effect of neuroleptic treatment (drugs that block dopamine function, and more recently in the atypical form also block serotonin function) which has been the primary treatment for schizophrenia, and the other psychoses. It was suggested that the symptoms of the disorder were due to a dopaminergic supersensitivity brought on by years of treatment with dopamine-blocking drugs. More recent studies have suggested that lifelong reduced dopamine synthesis due to chronically higher levels of plasma Phe may increase the vulnerability to the development of TD. These drugs are also used in the treatment of mood and personality disorders. Persons with mood disorders have been shown to have a particular vulnerability to the development of TD when treated with neuroleptics. All three groups of these disorders are believed to at least partially respond to treatment with medications that regulate amine neurotransmitter synthesis.
A summary of the studies which form the core of the Phe metabolic model are as follows:
1. The association of hyperphenylalanemia with TD (Study 1). A point prevalence study of TD (n=211) in a mentally retarded population showed that phenylketonuria, a severe hyperphenylalaninemia, was a significant risk factor for the presence of TD. These data suggested that high plasma Phe was associated with the development of TD (Richardson, M. A. et al., xe2x80x9cThe prevalence of tardive dyskinesia in a mentally retarded populationxe2x80x9d Psychopharmacol. Bull., 1986; 22: 243-249).
2. Protein challenge given to schizophrenic men with and without TD. A dietary challenge in the form of a high-protein meal (Phe=3.6% of total protein, branched chain amino acids (BCAA=19.6% of total protein) was administered to 53 male schizophrenics (Study 2). The findings were:
(a) Alteration of protein kinetics in men with TD. Data analyses established that the post-challenge plasma Phe level and post-challenge Phe/large neutral amino acid ratio were significantly higher in patients with TD and were significant predictors of TD status in male schizophrenic patients, independent of age. The ratio corrects for the competition of the other large neutral amino acids with Phe at the blood-brain barrier (Richardson, M. A. et al., xe2x80x9cThe plasma phenylalanine/large neutral amino acid ratio: a risk factor for tardive dyskinesiaxe2x80x9d Psychopharmacol. Bull., 1989; 25: 47-51; Richardson, M. A. et al., Comment on xe2x80x98The ratio of plasma phenylalanine to other large neutral amino acids is not a risk factor for tardive dyskinesiaxe2x80x99 J. Psychopharm., 1993; 7: 2; Richardson, M. A. et al., xe2x80x9cTardive dyskinesia and phenylalanine metabolism: risk-factor studiesxe2x80x9d Ch. 13, in: Yassa, R. et al., eds. Neuroleptic-Induced Movement Disorders. Cambridge: Cambridge University Press, 1997: 161-174; Richardson, M. A. et al., xe2x80x9cPlasma phenylalanine: A measure of tardive dyskinesia vulnerability in schizophrenic malesxe2x80x9d Chap.7, in: Richardson, M. A., eds. Amino Acids in Psychiatric Disease. Washington, D.C.: American Psychiatric Press, 1990: 143-160).
(b) Unexpected total remission of TD symptoms. More than half of the patients with TD had either a complete remission of their TD symptoms or a minimum 50% decline in symptoms two hours after the protein meal. Plasma analyses demonstrated that there were significantly lower Phe/large neutral amino acid and tyrosine/large neutral amino acid ratios and significantly higher branched chain amino acid/large neutral amino acid ratios in the group of patients who had a remission, as compared with those who did not. The authors hypothesized that the protein challenge comprised of high branched chain amino acid content, may have reduced the availability of Phe and tyrosine to the central nervous system, effecting the dramatic symptom remission (Richardson, M. A. et al., xe2x80x9cPhenylalanine to serotonin to tardive dyskinesia: A new modelxe2x80x9d, Proceedings of the 2nd International Symposium on Serotonin, from Cell Biology to Pharmacology and Therapeutics, Houston, Tex. Sep. 15-18, 1992; Richardson, M. A. et al., xe2x80x9cA dietary intervention decreases tardive dyskinesia symptomsxe2x80x9d Am. Psychiatr. Assoc., 149th Annual Meeting, New York, N.Y. 1996; 0:194).
3. Alteration of Phe kinetics in men with ID (Study 3). The study hypothesis, that TD would be associated with significantly higher plasma Phe indices (absolute plasma Phe level, plasma Phe/large neutral amino acid ratio) two hours after a Phe challenge, was verified for the male participants in the study (N=209; total N=312). The altered kinetics of Phe in men with TD indicated that there was a greater availability of Phe to the brains of these men. These data suggest that the disorder may be related to the effects of this greater availability. Such effects could be the direct neurotoxic effects of Phe and its metabolites and/or the modulating effects of these compounds on the synthesis of the monoamine neurotransmitters (Richardson, M. A. et al., xe2x80x9cTardive dyskinesia and phenylalanine metabolism: risk-factor studies. Ch. 13, in: Yassa, R. et al., eds. Neuroleptic-Induced Movement Disorders. Cambridge; Cambridge University Press, 1997: 161-174; Richardson, M. A. et al., xe2x80x9cPhenylalanine metabolism: Sex and Age issuesxe2x80x9d Schizophrenia Research, 1996; 18: 149; Richardson M A, Reilly M A, Read L L, Flynn C J, Suckow R F, Maher T J, Sziraki I: Phenylalanine kinetics are associated with tardive dyskinesia in men but not in women. Psychopharmacology, in press).
4. Treatment of TD with a dietary supplement of the BCAA (Study 4). Clinical trials were conducted to determine whether a drink containing exactly the same amount and proportion of the BCAA as was in the protein challenge meal in Study 2 (given three times a day for two weeks) would decrease TD symptoms (Richardson M A, Bevans M L, Weber J B, Gonzalez J J, Flynn C J, Read L L, Suckow R F, Maher T J: Branched chain amino acids decrease tardive dyskinesia symptoms. Psychopharmacology, in press; (Richardson, M. A. et al., xe2x80x9cA dietary intervention decreases tardive dyskinesia symptomsxe2x80x9d Am. Psychiatr. Assoc., 149th Annual Meeting, New York, N.Y. 1996; 0: 194; Richardson, M. A. et al., xe2x80x9cTD symptom decreases with regulation of plasma large neutral amino acids. Schizophrenia Research 1997; 24:272; Richardson, M. A. et al., xe2x80x9cTD symptom decreases with regulation of plasma large neutral amino acidsxe2x80x9d Abstracts, 16th International Congress of Nutrition, Montreal, Canada 1997; 0:42). A statistically significant decrease in the level of TD symptoms was observed for the sample. The symptom changes were also clinically significant in that 6 of the 9 subjects had symptom decreases of at least 58%, with all subjects having a decrease of at least 38%. Branched chain amino acid administration increased plasma branched chain amino acid concentrations and branched chain amino acid/large neutral amino acid ratios, and decreased plasma levels and large neutral amino acid ratios of Phe, tyrosine, and tryptophan. Analyses indicated a strong significant correlation between the percent increase in the plasma branched chain amino acid values at the first administration and the percent improvement in TD over the trial in eight of the nine subjects. The study findings suggested that the decrease in TD symptoms was modulated by a decrease in the brain uptake of Phe, a decrease in neurotransmitter synthesis, and/or the increase of the branched chain amino acid and decrease of aromatic amino acids in the periphery.
5. Placebo-controlled trial for the BCAA as a treatment for TD in adult men (Study 5). A placebo-controlled trial of the BCAA drink for the treatment of TD in men was designed to eliminate ineffective doses. For a response criteria of 50% decrease in symptoms, no responders were observed at the placebo dose, or at a low dose. A 17% rate of responders was observed at the mid-dose, and a 50% rate of responders in the high dose group. The trend in these data suggested a significant active versus placebo group response.
6. Pilot trial of the BCAA in the treatment of dyskinesias in neuroleptic-treated children and adolescents 9 (Study 6). Substantial decreases in symptoms for dyskinesias were seen in five out of six adolescents treated for two weeks. The decreases ranged from 37% to 68%. Two adolescent boys went into roll-on treatment periods. The percent decrease in symptoms was accelerated in the longer treatment periods. One subject who showed a 50% decrease after two weeks of treatment showed a 94% decrease after 31 days, and a second subject who showed a 54% decrease after two weeks of treatment showed a 74% decrease after 54 days of treatment.
7. DNA analysis of the PAH gene in psychiatric patients and controls (Study 7). The association between TD and diminished Phe kinetics in combination with the reduction of TD symptoms following a medical food product, led to the screening of the PAH gene in patients with psychotic, mood and personality disorders both with and without TD. The study hypothesis was that PAH variants may cause modest but significant changes in PAH activity that result in differences in Phe kinetics following a dietary Phe dose. It was further hypothesized that PAH variants may also have long term effects including predisposition to TD and psychotic disorders The findings of this study comprise the object of the invention, which is presented in the Field of Invention, and described in the Detailed Description and Examples 1-7.
Central to the present invention is the discovery of (a) point mutations and polymorphisms of the PAH gene that differ from the predominant wild-type PAH gene, in persons diagnosed with Psychotic, Mood or Personality Disorders, (b) significant associations between these sequence altered forms of the PAH gene and the presence of one of the Psychotic Disorders, Schizophrenia, and (c) significant associations between the sequence altered forms of the PAH gene and enzyme, and experimental plasma responses indicative of reduced function of the enzyme.
The invention includes the detection of sequence altered forms of the PAH gene, in order to define pathophysiological subtypes of the Psychotic, Mood or Personality Disorders, particularly Schizophrenia that would respond to treatment or prevention with a class of amino acid products already developed and patented by the Inventor. The epidemiology of these PAH variants in the Inventor""s work suggests that certain of these variants will be particularly relevant for persons of African ethnicity. These subtypes can be defined by dual markers; one, the presence of PAH gene variants, and/or, two, an experimental plasma reponse to a Phe dose defining an altered function of the PAH enzyme. For persons already afflicted with Psychotic, Mood or Personality Disorders these data will allow for the definition of a pathophysiological subtype that will respond to treatment with one or more of a range of treatments. For their relatives, without psychiatric disease, these data will allow for detection of those relatives who have an increased susceptibility to the development of such psychiatric disease, and thus would be candidates for preventative treatment with the same range of treatments as.
The invention includes a method for detecting the presence of DNA encoding variants of the PAH gene, which comprises isolating DNA and amplifying a region of the DNA that encodes a nucleotide substitution, wherein a nucleotide substitution indicates the presence of a mutation or a polymorphism. The amplification step comprises a polymerase chain reaction involving specific primers complementary to regions throughout the PAH gene. Primer pairs are designed for either DGGE or SSCP methods of gradient gel filtration.
The present invention is related to the discovery of two mutations and ten polymorphisms on the PAH gene in a group of 124 psychiatric patients carrying the diagnoses of Psychotic, Mood or Personality Disorders. The PAH enzyme catalyzes the conversion of the large neutral amino acid, Phe to tyrosine, which is the rate limiting step in the catabolism of Phe. The brain is highly sensitive to levels of Phe and deficiencies in the PAH enzyme can result in excess levels of Phe (hyperphenylalanemia) with a wide range of phenotypes, the most serious being the mental retardation of classical PKU. Phe, as an indirect precursor of the neurotransmitters dopamine and noradrenaline, supports their synthesis at low plasma concentrations while at higher concentrations it inhibits the synthesis of these two neurotransmitters and that of a third, serotonin. Thus plasma Phe levels play an important role in modulating neurotransmitter synthesis. The amine neurotransmitters (dopamine, noradrenaline, and serotonin) have long been considered to be relevant to the pathophysiology of the Psychotic, Mood and Personality Disorders, and the regulation of these neurotransmitters has been the goal of the medications developed to treat most of these disorders.
One of the PAH mutations (K274E), found in the Inventor""s study, is novel, while the other mutation (A403V) is known to cause hyperphenylalanemia. Four polymorphisms are novel (IVS6nt-7, N133N, L321L, N426N) while the remaining six (IVS2nt19, IVS3nt-22, Q232Q, V245V, L385L, Y414Y) were already known. The K274E and the L321L variants were significantly associated with Schizophrenia (a Psychotic Disorder) and with each other, while the IVS2nt19 polymorphism showed a trend to a significant association with the disorder.
Ethnic considerations, particularly African-American status, were important in the data set since (a) all persons with the K274E and L321L variants (which were associated with schizophrenia) were African-Americans, (b) three of the five novel variants (K274E, L321L, N426N) was significantly associated with being African-American, and (c) for one of the other novel variants (IVS6nt-7) all Schizophrenics with this variant were African-American. The one person in the data set with the A403V mutation was Caucasian and schizophrenic. Caucasian ethnic status was significantly associated with three known polymorphisms (IVS3nt-22, Q232Q, V245V). Ethnic status has been a critical variable in uncovering the genetic background to disease expression, for instance in the case of Alzheimer""s Disease, the APOE-e4 allele is consistently associated with a greater risk for the disease in Caucasians but not in African-Americans. In Schizophrenia, significant differences have been seen between those of African and European descent in heterozygosity and marker allele frequency, leading investigators to separate these groups for genetic analysis.
Taking a unique approach to the analysis of the genetics involved in the Phe metabolic pathway, all subjects who participated in the DNA study performed by the Inventor were exposed to a physiological experimental situation to determine the efficiency of PAH activity. Phe, the amino acid catabolized by PAH, was given to subjects in a 100 mg/kg body weight dose. Blood samples for large neutral amino acid analysis were collected fasting and two hours after the Phe dose administration in order to be able to test the kinetics of Phe. Fasting blood samples were also collected for analysis of neopterins and biopterins which are indicators of BH4 synthesis and utilization. BH4 is critical for amine neurotransmitter synthesis in its capacity as a cofactor for PAH (Phe to tyrosine), tyrosine hydroxylase (tyrosine to dopamine), and tryptophan hydroxylase (tryptophan to serotonin).
Information which is critical to the utilization of the Invention and unique to the field of the genetics of Psychotic, Mood and Personality Disorders was defined by the analysis of the results of the plasma assays performed. Patients with the K274E mutant protein were seen to have a significantly reduced ability to convert the ingested Phe to tyrosine, and a significantly lower neopterin/biopterin ratio (indicative of an increase in BH4 synthesis). Persons with the L321L polymorphism also showed significantly reduced ability to convert the ingested Phe to tyrosine. The person with the A403V mutation showed a similar metabolic profile but to a more pronounced degree which is consistent with her hyperphenylalanemic status.
In Examples 8 and 9 the PAH K274E mutation is used as a marker to define a subtype of schizophrenia and susceptibility to schizophrenia in those of African-American ethnicity. The subtype of schizophrenia defined by the presence of this mutation will be one whose symptoms respond to treatment that lowers the availability of Phe to the brain. For first and second degree relatives of schizophrenics, the presence of the PAH K274E mutation and/or altered plasma values of Phe, tyrosine, biopterin and neopterin will define persons who may be susceptible to the development of schizophrenia.
The Inventor has patented a medical food product composed of the branched chain amino acids (BCAA) (U.S. Pat. No. 5,393,784) for use in TD. The BCAA formulation that has been used is valine: isoleucine: leucine at a ratio of 3:3:4. This product has been clinically proven to decrease Phe availability to the brain. The BCAA product can be used to treat schizophrenic symptoms in those persons with the K274E mutation. Additionally, the BCAA (in the above ratio) can be used to prevent the development of Schizophrenia in their first and second degree relatives.
Elaboration of methods proposed in Example 8 are as follows:
1. DGGE Kit. Kits will be useful for the detection of sequence variants in the PAH gene. The kit will be comprised of a set of 14 primer oligonucleotides, and an information sheet containing the primer sequences and the wild-type phenylalanine hydroxylase gene sequence.
Sequence variants will be identified following extraction of DNA from blood cells or other biological samples collected from subjects for screening. Extraction of the DNA can be performed by methods known in the field of art. The extracted DNA will be subjected to PCR amplification of fragments covering the PAH variants of Example 1, followed by denaturing gradient gel electrophoresis (DGGE). The kit will provide the primers for the PCR amplification that will correspond to SEQ ID Nos. 3, 4, 7, 8, 11-14, 17, 18, 21-24. The PCR-amplified DNA fragments will be fractionated on polyacrylamide gels with urea and/or formamide gradients. After electrophoresis, band patterns will be visualized using a phosphoimager or other visualization system. The primer sequences and methods have been previously described (Guldberg P and Guttler F, xe2x80x9cA simple method for identification of point mutations using denaturing gradient gel electrophoresisxe2x80x9d, Nucleic Acids Res., 1993, 21: 2261-2; Guldberg P et al., xe2x80x9cMolecular analysis of phenylketonuria in Denmark: 99% of the mutations detected by denaturing gradient gel electrophoresisxe2x80x9d, Genomics, 1993, 17 141-6; Guldberg et al., xe2x80x9cMutational spectrum of phenylalanine hydroxylase deficiency in Sicily: implications for diagnosis of hyperphenylalanemia in Southern Europexe2x80x9d, Hum Mol Genet, 1993, 2: 1703-1707; Guldberg P et al., xe2x80x9cPhenylalanine hydroxylase gene mutations in the United States: report from the Maternal PKU Collaborative Studyxe2x80x9d, Am J. Hum Genet., 1996, 59: 84-94).
2. SSCP kit. The single stranded conformation polymorphism (SSCP) method is the preferred method of detection for known gene sequence variants at the single base-pair level in a clinical laboratory environment (Orita-M et.al, xe2x80x9cDetection of polymorphisms of human DNA by gel electrophoresis as single-stranded conformation polymorphismsxe2x80x9d, Proc Natl Acad Sci USA, 1989, 86: 2766-2760). For detection of the PAH gene sequence variants described in this Invention, a kit will be comprised of a set of 24 primer oligonucleotides corresponding to SEQ ID Nos. 39-62. Also included will be PCR products from control individuals who are homozygous and heterozygous for each PAH variant targeted, to serve as electrophoretic mobility standards. In addition the kit will include an information sheet containing the primer sequences and a list of the PAH variant sequences detected by the kit.
3. Immunological detection. Diagnostic kits used to detect the K274E mutation by immunological detection may include a solid support capable of binding the PAH/K274E protein isoform, and further include one or more antibodies which are allele-specific and whose binding to the specific allele can be detected by the generation of a signal or the detection of a label. Other versions of a diagnostic kit include a kit comprising an allele-specific antibody attached to a solid support, capable of binding an antigen which is the PAH/K274E allele, and a means for detection of the antibody-antigen complex.
Alternative uses of the Invention to those proposed in Examples 8 and 9 are as follows:
1. The L321L variant is significantly associated with schizophrenia and with the presence of the K274E mutation, and persons with this mutation show a significantly reduced ability to convert ingested Phe to tyrosine. The presence of this variant can also be used to define a subtype of persons with Schizophrenia whose symptoms may respond to treatment with the BCAA. This variant can also be used to define first and second degree relatives of schizophrenics who will then be tested as described in Example 9 by administration of a 100 mg/kg dose of Phe. Post dose plasma values of Phe, and tyrosine, and percent change values from fasting to post dose will be studied for both amino acids. If test results are within the parameters listed in Example 9 then these relatives may be defined as susceptible to developing Schizophrenia. The definition may be used to qualify these relatives as candidates for prophylactic treatment with the BCAA.
2. For the three other novel polymorphisms (IVS6nt-7, N133N, N426N), patients with these variants can be tested as described in Example 9 by administration of a 100 mg/kg dose of Phe. Post dose plasma values of Phe, and tyrosine, and percent change values from fasting to post dose can be studied for both amino acids. If test results are within the parameters listed in Example 9 then these patients may be candidates for treatment with the BCAA.
3. The known IVS2nt19 polymorphism showed a trend for an association with schizophrenia, but showed a significantly lower post Phe dose level of Phe and a significantly lesser response to a 100 mg/kg Phe dose than in patients without the polymorphism. This suggests a dysregulation related to the psychosis that might respond to the use of administered Phe. The Inventor has patented the use of Phe in movement disorders (U.S. Pat. No. 5,670,539; allowed application U.S. application Ser. No. 08/545,095).
4. The presence of the A403V mutation in patients with schizophrenia may serve as a marker for a BCAA treatment response (aimed at reducing Phe availability to the brain). The first and second degree relatives of a person with this mutation and schizophrenia should be screened for the presence of this mutation. If present, they should then be tested to see if they have Phe and tyrosine plasma values two-hours subsequent to a 100 mg/kg Phe dose comparable to those seen in this patient (see Example 5.) or within the parameters presented in Example 9, then these relatives may be defined as susceptible to developing Schizophrenia. The definition may be used to qualify these relatives as candidates for prophylactic treatment with the BCAA.
5. Where a linkage between any of the two mutations and ten polymorphisms, and the presence of schizophrenia in African-American pedigree studies (See Example 8) has been demonstrated, then those variants can be used to define both a subtype of schizophrenia in patients with the disorder and susceptibility to schizophrenia in relatives who have not yet shown symptoms of the disorder. Both these groups would be candidates for treatment with the BCAA.
6. Any persons with Psychotic, Mood and Personality Disorders who are shown to have any of these two mutations and ten polymorphisms in the PAH gene can be tested as described in Example 9 by administration of a 100 mg/kg dose of Phe. Post dose plasma values of Phe, and tyrosine, and percent change values from fasting to post dose can be studied for both amino acids. If test results are within the parameters listed in Example 9 then treatment with BCAA can be offered. For disorders such as Major Depressive Disorder where deficits in tyrosine may be seen, consideration will be given to treatment with tyrosine. The Inventor has patented the use of tyrosine in movement disorders (U.S. Pat. No. 5,670,539; allowed application U.S. application Ser. No. 08/545,095).
7. Persons with Psychotic, Mood and Personality Disorders, in the absence of (a) the determinations of PAH variants, (b) plasma responses to a Phe dose in Phe and tyrosine levels, or (c) biopterin and neopterin levels, would be seen as candidates for treatment with the BCAA if the treatment mechanism of action of these amino acids is considered appropriate for their disorder.
Further details regarding this invention are presented but are not limited to the Examples.